Rapid, sensitive, specific, and cost-effective biochemical assays are needed for a variety purposes. For example, the recent emergence of Sudden Acute Respiratory Syndrome (SARS) and national security bio-threats indicate the need to identify infectious agents or toxins for appropriate therapeutic intervention. It would be valuable to be able to simultaneously detect and identify in clinical samples a broad range of infectious agents such as the corona virus responsible for SARS. Another example is the need for more sensitive, specific, accurate, reliable, easy to use, and inexpensive research tools to measure RNA expression patterns of small cell numbers, such as might be obtained from a laser-capture microdissection.
One primary challenge for bio-defense and diagnostic applications is the early detection of infections, which typically requires increasing assay sensitivity. Presently, the most sensitive and widely used molecular diagnostic methods are based on real-time Polymerase Chain Reaction (PCR) methods such as TaqMan® for amplifying pathogennucleic acids. However, these methods suffer from several limitations:
1) The cost of the assays and amount of sample needed are often too prohibitive to run large numbers of assays against a patient sample.
2) The assays amplify but do not concentrate nucleic acids. For example, if there are 10 copies of SARS RNA in a patient sample, performing assays against 20 viral sequences involves a significant risk of obtaining a false negative test result.
3) Multiplexing numerous assays is quite difficult due to the need to harmonize reaction conditions and separate results into different optical channels. A typical problem in PCR multiplexing is the competition between the many primers.
4) Screening for potential bio-terrorism agents tend to be done only at the state and federal level, and not at the clinic or local level. That is because even with an assay that was 99.9% accurate, the numerous false positives that would occur with widespread screening would result in unreasonable expense as well as economic and political disruption. Thus, there is a need for a great increase in the reliability of such tests.
Some of these problems can be addressed by using parallel microfluidic assay arrays. One example of such an array is the Living Chip™ marketed by Biotrove, Inc. of Woburn, Mass. In function and purpose, the Living Chip™ is similar to 96- and 384-well microtiter plates currently used in high-throughput screening and diagnostics. However, the approximately 35 nl sample volume held by each sample well in the Living Chip™ is roughly 2000 times less than that in a 96-well plate, and 200 times less than a 384-well plate.
FIG. 1 shows a cut away view of a typical microfluidic sample array of through-holes. Such an array is described, for example, in U.S. Pat. No. 6,387,331 and U.S. Patent Application 20020094533, the contents of which are incorporated herein by reference. The sample array 10 includes a sheet of material 14 having a pair of opposed surfaces and a thickness. The sheet of material 14 may be a platen, otherwise referred to herein as a chip, and may made of, for example, conductive silicon, or other types of rigid materials, such as metal, glass, or plastic. A large number of through-holes 12 run through the thickness from one of the surfaces 14 to the other opposing surface (not shown).
The sample array 10 typically may be from 0.1 mm to more than 10 mm thick; for example, around 0.3 to 1.52 mm thick, and commonly 0.5 mm. Typical volumes of the through-holes 12 may be from 0.1 picoliter to 1 microliter, with common volumes in the range of 0.2-100 nanoliters, for example, about 35 nanoliters. Capillary action or surface tension of the liquid samples may be used to load the sample through-holes 12. For typical chip dimensions, capillary forces are strong enough to hold liquids in place. Chips loaded with sample solutions can be waved around in the air, and even centrifuged at moderate speeds without displacing samples.
To enhance the drawing power of the through-holes 12, the target area of the receptacle, interior walls 13, may have a hydrophilic surface that attracts a liquid sample. It is often desirable that the surfaces be bio-compatible and not irreversibly bind biomolecules such as proteins and nucleic acids, although binding may be useful for some processes such as purification and/or archiving of samples. Alternatively, the sample through-holes 12 may contain a porous hydrophilic material that attracts a liquid sample. To prevent cross-contamination (crosstalk), the exterior planar surfaces 14 of chip 10 and a layer of material 15 around the openings of sample through-holes 12 may be of a hydrophobic material. Thus, each through-hole 12 has an interior hydrophilic region bounded at either end by a hydrophobic region.
The use of through-holes 12, as compared to closed-end well structures, reduces the problem of trapped air inherent in other microplate structures. The use of through-holes together with hydrophobic and hydrophilic patterning enables self-metered loading of the sample through-holes 12. The self-loading functionality helps in the manufacture of arrays with pre-loaded reagents, and also in that the arrays will fill themselves when contacted with an aqueous sample material.
It has been suggested that such arrays can be utilized for massively parallel PCR analysis of a given sample. For example, International Patent Application WO 01/61054 (incorporated herein by reference) suggests that sample probes and PCR reagents can be dried onto the walls of the sample wells. One problem that has been observed with this approach is that when the array is immersed in a sample liquid to load the through-holes, the dried probes and reagents can dissolve and float away out of the sample wells that they were loaded in.
Additionally, with PCR, a series of heating and cooling cycles is used to replicate a small amount of DNA into a much larger amount. Thermal cyclers are devices that generate such a series of heating and cooling cycles. Current thermal cycling approaches are not well suited for thermal cycling of sample arrays such as the one shown in FIG. 1. Unlike standard microtiter plates having closed-end storage wells, the sample arrays with through-holes cannot be simply set on a temperature controlled thermal block because some or all of the samples can be wicked out of their storage channels onto the supporting plate. Nor are such through-holes suitable for immersion in a temperature controlled circulating fluid because the fluid would be free to enter the hole openings and could mix with or extract the contents of the through-holes. Also, if fluid flow is used to produce a temperature change, pressure differences within the fluid can cause the sample to leave the through-holes.
The great densities and small volumes for the through-holes 12 of the sample array pose further challenges to implementing various complex assays in such systems. Such challenges include risks of (i) chemical and physical interactions between adjacent through-holes, (ii) loss of sample below an amount permitting reliable assay, (iii) non-uniformity of assay from through-hole to through-hole, so as to impair the reliability of assays using such systems, (iv) the ability to load samples into the array, and (v) inhibitory or otherwise unfavorable interactions between the surfaces of the array and the reagents or samples in the reactions.